Bacteria sense their environments within diverse locations in the host and respond by appropriately altering their cell surface structures to avoid immunity and promote colonization. The molecular interactions with the host that necessitate signal transduction by bacteria and the cues they utilize to respond appropriately are not well defined. Haemophilus influenzae provides a genetically tractable model to examine this aspect of pathogenesis. H. influenzae efficiently colonizes the human nasopharyngeal mucosa in an apparently commensal mode. Its subsequent spread to other sites frequently causes disease including otitis media, exacerbations of chronic obstructive pulmonary disease, and community acquired pneumonia. In addition, some strains invade the bloodstream, causing bacteremia and meningitis. The mechanisms that H. influenzae uses to negotiate these disparate environments are not well understood. Our results indicate that H. influenzae senses and responds to redox conditions, such as oxygen availability, to control critical virulence properties including the structure of its lipooligosaccharide (LOS) and resistance to hydrogen peroxide. The outermost structure of LOS is composed of a variable set of carbohydrates and other modifications collectively termed the outer core. LOS structures are essential for H. influenzae to colonize the host, but also represent targets of innate and adaptive immunity. One such structure, phosphorylcholine (PC), contributes to pathogenesis by promoting adherence to host cells, yet is also a target for host immune effectors such as innate antibodies and C-reactive protein. We demonstrated that modification of the LOS with PC is increased under low oxygen conditions and diminished in response to high oxygen {Wong, 2005 #6543}. These and other results support the view that redox conditions provide environmental cues used by H. influenzae during stages of pathogenesis in which it encounters sites such as the mucosal epithelium, bloodstream, and lungs that differ with respect to both oxygen availability and the presence of antimicrobial defenses. Consistent with this hypothesis, the redox responsive regulator, ArcA, is required for pathogenesis in animal models. ArcA regulates several virulence phenotypes including LOS outer core modification, serum resistance, and resistance to oxidative stress. These virulence properties are likely critical during the transition from the airway surface to interstitial invasion of epithelial tissue or during late stages of respiratory infection in which levels of complement and numbers of activated phagocytes are increased in comparison to those at the normal airway surface. To understand the mechanisms by which virulence factor regulation in response to environmental signals contribute to the ability of H. influenzae to evade and resist host immune responses, we plan to achieve the following aims: Aim#1. Identify and characterize the regulation of H. influenzae surface structures responsible for recognition by bactericidal components of serum. Our preliminary results provide evidence that surface structures involved in serum resistance of H. influenzae are regulated in response to redox conditions of growth. We will use genetic and biochemical approaches to identify genes responsible for resistance and determine the mechanism of regulation of relevant cell-surface structures. Aim#2. Delineate host defense mechanisms that target regulated bacterial surface structures. Complement mediated lysis is the major mechanism acting on H. influenzae in serum bactericidal assays in vitro and correlates with virulence in vivo. We will investigate molecular interactions with components and modulators of complement that are influenced by regulation of bacterial cell surface structures. Complement components have diverse roles in anti-microbial immunity, and understanding the step that is inhibited will provide insight into the mechanism by which regulation of complement resistance influences pathogenesis. Aim#3. Evaluate the role of regulation in response to redox conditions during H. influenzae pathogenesis. LOS outer core structures are essential in immune evasion and we have detected regulation of these structures in response to environmental signals. Bacterial mutants designed to address the role of LOS gene regulation will be evaluated in animal models of infection. Immune signaling and effector pathways will be examined to define clearance mechanisms that H. influenzae is able to avoid by modulating its surface structures.